ALDA-1 treatment and prevention of pulmonary disease

ABSTRACT

Methods of treating or preventing allergic or pulmonary diseases characterized by endothelial dysfunction with Alda-1 are presented. Treatment of pulmonary endothelial cells subjected to hyperoxia with Alda-1 showed an increase in ALDH2 activity and expression. Treatment with Alda-1 also illustrated a decrease in oxidative stress, a decrease in reactive oxygen species (ROS), a decrease in apoptosis, a decrease in inflammation and an enhancement of mitochondrial membrane potential.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to currentlypending U.S. Nonprovisional application Ser. No. 16/282,966, entitled“ALDA-1 Treatment and Prevention of Pulmonary Disease”, filed Feb. 22,2019, which claims priority to U.S. Provisional Patent Application No.62/634,539, entitled “ALDA-1 Shields Endothelial Cells Against OxidativeStress Via Activation of ALDH2,” filed Feb. 23, 2018, the contents ofeach of which is incorporated by reference into this disclosure.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under Grant No. HL105932awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates, generally, to allergic and/or pulmonary diseasesand the treatment of allergic and/or pulmonary diseases. Morespecifically, it relates to treating endothelial dysfunction and changesin vascular permeability.

BACKGROUND OF THE INVENTION

Endothelial dysfunction and changes in vascular permeability are keyevents in many allergic diseases including edema and chronic obstructivepulmonary disease (COPD). Mitochondrial dysfunction causes elevation ofreactive oxygen species (ROS). ALDH2 (aldehyde dehydrogenase 2) is knownto be an effective combatant against mitochondrial dysfunction. Humancarriers possessing an inactive ALDH2 variant have been found to be moresusceptible to COPD and asthma. However, information pertaining to theALDH2 interactome is limited.

Aldehyde dehydrogenases (ALDH) constitute a family of enzymes that playa critical role in detoxification of various cytotoxic xenogenic andbiogenic aldehydes. ALDH is a key enzyme in fructose, acetaldehyde andoxalate metabolism and represents a major detoxification system forreactive carbonyls and aldehydes. There are at least 19 members/isozymesof the ALDH family, where the various isozymes may exhibit differentsubstrate specificity and/or cellular localization relative to othermembers of the family.

Cytotoxic aldehydes derive from a variety of sources. For example,environmental (external) sources of aldehydes include those that resultfrom ethanol consumption, from consumption of food sources, fromingestion of hazardous materials such as vinyl chloride, pesticides,herbicides, or from inhalation of hazardous materials such as thosefound in cigarette smoke, or industrial pollution. Aldehydes, that maybe cytotoxic, can also be produced biologically (e.g., endogenously),e.g., as a result of oxidative stress such as occurs in ischemia,irradiation, or metabolism or bioconversion of cellular precursors suchas neurotransmitters and drugs. Accumulation of cytotoxic levels ofaldehydes, and/or defects in the ALDH enzyme, has been implicated in avariety of diseases and conditions, or in increased risk of diseasedevelopment. The range of implicated diseases includes neurodegenerativediseases, aging, cancer, myocardial infarction, stroke, dermatitis,diabetes, cataracts, and liver diseases.

Accordingly, what is needed is a therapy targeted towards activation ofALDH2. However, in view of the art considered as a whole at the time thepresent invention was made, it was not obvious to those of ordinaryskill in the field of this invention how the shortcomings of the priorart could be overcome.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in asimplified form. These concepts are described in further detail in thedetailed description of example embodiments of the disclosure below.This summary is not intended to identify key features or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter.

The present disclosure provides compounds that function as modulators ofaldehyde dehydrogenase 2 (ALDH2) enzymatic activity, as well ascompositions and formulations comprising the compounds. The presentdisclosure provides therapeutic methods involving administering asubject compound, or a subject pharmaceutical composition.

Embodiments disclosed herein include methods of preventing endothelialcell injury from hyperoxic injury. For example, in accordance with oneembodiment, administering a therapeutically effective amount of Alda-1and a pharmaceutically acceptable excipient, reduces damage toendothelial cells from oxidative stress and activates ALDH2.

Alda-1 rescues mitochondrial membrane potential; decreases cytochrome Crelease; suppresses mitochondrial reactive oxygen species production;and preserves mitochondria.

The invention includes a method of treating pulmonary diseasecharacterized by endothelial dysfunction, the method includes the stepsof administering a therapeutically effective amount of Alda-1 to asubject suffering from the pulmonary disease characterized byendothelial dysfunction. The Alda-1 activates ALDH2 wherein the Alda-1reduces damage to endothelial cells from oxidative stress.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a series of MitoSOX images.

FIG. 2 is a series of images depicting JC1 staining of cells innormoxia, hyperoxia, and hyperoxia with Alda-1 at 24 hours.

FIG. 3 is a series of images depicting JC1 staining of cells innormoxia, hyperoxia, and hyperoxia with Alda-1 at 48 hours.

FIG. 4 is a series of images depicting certain results of the presentinvention from normoxia, hyperoxia, and hyperoxia with Alda-1 oncytochrome C, beta actin, and beclin at 24 hours and 48 hours.

FIG. 5 is an image depicting a graph that representing the whole cellprotein lysate for human microvascular endothelial cells under differentconditions subjected to ELISA to evaluate the levels of 4HNE contentduring the Normoxia (NO), hyperoxia (HO), hyperoxia+Alda1 (HO+Alda-1)(20 μM) for 48 hours. The results are shown in mean±SEM.

FIG. 6A is an image depicting the western blot analysis of the wholecell lysate for human microvascular endothelial cells under differentconditions (n=3) to evaluate the levels of cytochrome C during normoxia,hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours. Equal amountsof protein loaded per lane (20 μg).

FIG. 6B is a graph depicting expression of Cytochrome C normalized toβ-actin and presented in arbitrary units. The results are shown inmean±SEM.

FIG. 7A is an image depicting the whole cell lysate for humanmicrovascular endothelial cells under different conditions (n=3)subjected to immunoblot to evaluate the levels of ALDH2 during normoxia,hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalizedagainst β-actin. The values indicate standard error mean (*P<0.05).Equal amounts of protein loaded per lane (20 μg).

FIG. 7B is a graph depicting expression of ALDH2 normalized to β-actinand presented in arbitrary units. The results are shown in mean±SEM.

FIG. 8A is an image depicting the whole cell lysate for humanmicrovascular endothelial cells under different conditions (n=3)subjected to immunoblot to evaluate the levels of Bax during normoxia,hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalizedagainst Beta actin. The values indicate standard error mean (*P<0.05).Equal amounts of protein loaded per lane (20 μg).

FIG. 8B is a graph depicting expression of Bax normalized to β-actin andpresented in arbitrary units. The results are shown in mean±SEM.

FIG. 9A is an image depicting the human microvascular endothelial cellsunder different conditions subjected to the JC1 staining to evaluate themitochondrial membrane potential during normoxia, hyperoxia andhyperoxia with Alda-1 (20 μM) for 48 hours. The values indicate standarderror mean (*P<0.05).

FIG. 9B is a graph depicting the Jc1 images from FIG. 9A quantifiedusing imageJ software with an intensity expressed in arbitrary units.The results are shown in mean±SEM (Magnification=200×).

FIG. 10 is a graph depicting the mitochondrial lysate for the humanmicrovascular endothelial cells under different conditions (n=3)subjected to enzymatic assay to evaluate the ALDH2 activity at 340 nmduring normoxia, hyperoxia and hyperoxia with Alda-1 (20 uM) for 48hours.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part thereof, and within which areshown by way of illustration specific embodiments by which the inventionmay be practiced. It is to be understood that other embodiments may beused, and structural changes may be made without departing from thescope of the present application. These embodiments are described insufficient detail to enable those of ordinary skill in the art topractice the present disclosure, and it is to be understood that otherembodiments may be utilized, and that structural, logical, andelectrical changes may be made within the scope of the disclosure.

It should be understood that any reference to an element herein using adesignation such as “first,” “second,” and so forth does not limit thequantity or order of those elements, unless such limitation isexplicitly stated. Rather, these designations may be used herein as aconvenient method of distinguishing between two or more elements orinstances of an element. Thus, a reference to first and second elementsdoes not mean that only two elements may be employed there or that thefirst element must precede the second element in some manner. Also,unless stated otherwise a set of elements may comprise one or moreelements.

Any headings used herein should not be considered to limit the scope ofembodiments of the invention as defined by the claims below and theirlegal equivalents. Concepts described in any specific heading aregenerally applicable in other sections throughout the entirespecification.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the context clearly dictates otherwise.

In an embodiment, the current invention includes an activator of ALDH2,specifically Alda-1, that amplifies ALDH2 activity and attenuatesendothelial dysfunction in pulmonary diseases. Alda-1 also known asN-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamid has the empiricalformula C₁₅H₁₁Cl₂NO₃ with molecular weight 324.16. Alda-1 has structure:

As used herein, the term “aldehyde dehydrogenase” or “ALDH” refers to anenzyme that oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenicaldehyde, or an aldehyde produced from a compound that is ingested,inhaled, or absorbed) to its corresponding acid in an NAD+-dependent oran NADP+-dependent reaction. For example, ALDH oxidizes aldehydesderived from the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, that are produced as aresult of oxidative stress, or that are produced during normalmetabolism, e.g., conversion of retinaldehyde to retinoic acid. Anexample of a biogenic aldehyde is acetaldehyde produced as a product ofalcohol dehydrogenase activity on ingested ethanol. An aldehydedehydrogenase can also exhibit esterase activity and/or reductaseactivity.

The term “ALDH” encompasses ALDH found in the cytosol, in themitochondria, microsome, or another cellular compartment. The term“ALDH” encompasses ALDH found primarily in one or a few tissues, e.g.,cornea, saliva, liver, etc., or in stem cells and embryos. The term“ALDH” encompasses any of the known ALDH isozymes, including ALDH1,ALDH2, ALDH3, ALDH4, ALDH5, etc.

As used herein, the term “mitochondrial aldehyde dehydrogenase-2” or“ALDH2” refers to an enzyme that oxidizes an aldehyde (e.g., a xenogenicaldehyde, a biogenic aldehyde, or an aldehyde produced from a compoundthat is ingested, inhaled, or absorbed) to its corresponding acid in anNAD+-dependent reaction. For example, ALDH2 oxidizes aldehydes derivedfrom the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, or that are producedduring normal metabolism. Mitochondrial ALDH2 is naturally found inmitochondria.

The term “ALDH2” encompasses ALDH2 from various species. Amino acidsequences of ALDH2 from various species are publicly available. Forexample, a human ALDH2 amino acid sequence is found under GenBankAccession Nos. AAH02967 and NP-000681; a mouse ALDH2 amino acid sequenceis found under GenBank Accession No. NP-033786; and a rat ALDH2 aminoacid sequence is found under GenBank Accession No. NP-115792. The term“ALDH2” encompasses an aldehyde dehydrogenase that exhibits substratespecificity, e.g., that preferentially oxidizes aliphatic aldehydes.

The term “ALDH2” as used herein also encompasses fragments, fusionproteins, and variants (e.g., variants having one or more amino acidsubstitutions, addition, deletions, and/or insertions) that retain ALDH2enzymatic activity. Specific enzymatically active ALDH2 variants,fragments, fusion proteins, and the like can be verified by adapting themethods described herein. “ALDH2” includes an enzyme that convertsacetaldehyde into acetic acid, e.g., where the acetaldehyde is formed invivo by the action of alcohol dehydrogenase on ingested ethanol.

As used herein, “about” means approximately or nearly and in the contextof a numerical value or range set forth means±15% of the numerical. Inan embodiment, the term “about” can include traditional roundingaccording to significant figures of the numerical value.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range.

As used herein, the term “subject,” “patient,” or “organism” includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).Typical hosts to which an agent(s) of the present disclosure may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.

As used herein, “treat”, “treatment”, “treating”, and the like refer toacting upon a condition (e.g., endothelial dysfunction) with an agent(e.g., Alda-1) to affect the condition by improving or altering it. Theimprovement or alteration may include an improvement in symptoms or analteration in the physiologic pathways associated with the condition.The aforementioned terms cover one or more treatments of a condition ina patient (e.g., a mammal, typically a human or non-human animal ofveterinary interest), and includes: (a) reducing the risk of occurrenceof the condition in a subject determined to be predisposed to thecondition but not yet diagnosed, (b) impeding the development of thecondition, and/or (c) relieving the condition, e.g., causing regressionof the condition and/or relieving one or more condition symptoms.

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refers completely or partially preventing (e.g., about 50% ormore, about 60% or more, about 70% or more, about 80% or more, about 90%or more, about 95% or more, or about 99% or more) a condition or symptomthereof and/or may be therapeutic in terms of a partial or complete cureor alleviation for a condition and/or adverse effect attributable to thecondition.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” or “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and/oradjuvant that are useful in preparing a pharmaceutical composition thatare generally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use and/or human pharmaceutical use.“A pharmaceutically acceptable excipient, diluent, carrier and/oradjuvant” as used in the specification and claims includes one or moresuch excipients, diluents, carriers, and adjuvants.

The term “therapeutically effective amount” as used herein describesconcentrations or amounts of components such as agents which areeffective for producing an intended result, including attenuation ofendothelial dysfunction. Compositions according to the present inventionmay be used to effect a favorable change in endothelial dysfunction,whether that change is an improvement, relieving to some extent one ormore of the symptoms of the condition being treated, and/or that amountthat will prevent, to some extent, one or more of the symptoms of thecondition that the host being treated has or is at risk of developing,or a complete cure of the disease or condition treated.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Isolated compounds are at least about80%, at least about 90% pure, at least about 98% pure, or at least about99% pure, by weight. The present disclosure is meant to comprehenddiastereomers as well as their racemic and resolved, enantiomericallypure forms and pharmaceutically acceptable salts thereof.

The term “administration” or “administering” is used throughout thespecification to describe the process by which a composition comprisingAlda-1 as an active agent, are delivered to a patient or individual fortherapeutic purposes. The composition of the subject invention andmethodology in use thereof can be administered a number of waysincluding, but not limited to, parenteral (such term referring tointravenous and intra-arterial as well as other appropriate parenteralroutes), subcutaneous, peritoneal, inhalation, vaginal, rectal, nasal,or instillation into body compartments.

Administration will often depend upon the amount of compoundadministered, the number of doses, and duration of treatment. In anembodiment, multiple doses of the agent are administered. The frequencyof administration of the agent can vary depending on any of a variety offactors, such as attenuation of endothelial dysfunction and the like.The duration of administration of the agent, e.g., the period of timeover which the agent is administered, can vary, depending on any of avariety of factors, including patient response, etc.

The amount of the agent contacted (e.g., administered) can varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry, and the like. Detectablyeffective amounts of the agent of the present disclosure can also varyaccording to instrument and film-related factors. Optimization of suchfactors is well within the level of skill in the art, unless otherwisenoted.

“In combination with,” or “co-administration,” as used herein, in thecontext of administering a first compound and at least a secondcompound, refers to uses where, for example, the first compound isadministered during the entire course of administration of the secondcompound; where the first compound is administered for a period of timethat is overlapping with the administration of the second compound, e.g.where administration of the first compound begins before theadministration of the second compound and the administration of thefirst compound ends before the administration of the second compoundends; where the administration of the second compound begins before theadministration of the first compound and the administration of thesecond compound ends before the administration of the first compoundends; where the administration of the first compound begins beforeadministration of the second compound begins and the administration ofthe second compound ends before the administration of the first compoundends; where the administration of the second compound begins beforeadministration of the first compound begins and the administration ofthe first compound ends before the administration of the second compoundends. As such, “in combination” can also refer to regimen involvingadministration of two or more compounds. “In combination with” as usedherein also refers to administration of two or more compounds which maybe administered in the same or different formulations, by the same ofdifferent routes, and in the same or different dosage form type.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present disclosure calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present disclosure depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

Acute lung injury (ALI) is caused by lengthened exposure to hyperoxialeading to oxidative stress by crucially impairing the pulmonaryfunction. Mitochondrial dysfunction is a main event in hyperoxic acutelung injury. It is known that hyperoxia causes cellular damage and deathby impairing mitochondrial Aldehyde dehydrogenase 2 (ALDH-2) (1) byinducing oxidative stress. It is an object of the current invention toevaluate the effects of Alda-1 on lung endothelial cells duringhyperoxic injury.

Example 1—Alda-1-Mediated Modulation of ALDH2 is an Effective Target forRemediation of Endothelial Dysfunction Through Preservation ofMitochondria

Materials and Methods

Cells obtained were human microvascular endothelial cells (HMVECs). Cellculture medium was EBM-2 containing 10% FBS plus antibiotics. Assayswere MitoSOX, JC1, Histology, Western blot, and ALDH2 activity.Antibodies used were mouse anti-ALDH2 antibody, Cytochrome C, and Beclin(cell signaling).

HMVECs were seeded and, after reaching confluence, treated with DMSO andAlda-1. Cells were then exposed to normoxia or hyperoxia at differenttime points. After 24, 48, and 72 hours, cells were collected formitochondrial analyses.

Results

Cells treated with Alda-1 displayed elevated ALDH2 activity during bothnormoxia and hyperoxia. It was also observed that Alda-1 rescuedmitochondrial membrane potential, decreased Cytochrome C release, andsuppressed mitochondrial ROS production in HMVECs. See FIGS. 1-4.

FIG. 1 depicts MitoSOX images. FIG. 2 depicts cells at 24 hours whichare exposed to normoxia, hyperoxia, and cells treated with Alda-1. FIG.3 depicts cells at 48 hours which are exposed to normoxia, hyperoxia,and cells treated with Alda-1. FIG. 4 depicts certain results of thepresent invention. FIG. 4 depicts normoxia, hyperoxia, and the resultsof Alda-1 on hyperoxia cells on cytochrome C, Beclin, and Beta actin at24 hours and 48 hours.

Conclusion

The findings suggest that Alda-1-mediated modulation of ALDH2 is aneffective target for remediation of endothelial dysfunction throughpreservation of mitochondria. These results reveal a promisingtherapeutic approach to treating allergic diseases where endothelialdysfunction is a characteristic event.

Example 2—Prophetic

It is an object of the current invention to evaluate the effects ofAlda-1 on lung endothelial cells in vivo during hyperoxic injury.

Materials and Methods

Lung endothelial cells are treated with Alda-1 and a pharmaceuticallyacceptable excipient. Cells are then exposed to normoxia or hyperoxia atdifferent time points. After 24, 48, and 72 hours, cells are collectedfor mitochondrial analyses.

Results

Cells treated with Alda-1 display elevated ALDH2 activity during bothnormoxia and hyperoxia. It is also observed that Alda-1 rescuesmitochondrial membrane potential, decreases Cytochrome C release, andsuppresses mitochondrial ROS production in lung endothelial cells.

Example 3—Alda-1 Treatment In Vitro Diminishes the Accumulation of 4HNEand Reduces Oxidative Stress by Decreasing ROS During Hyperoxia

The inventors examined the capacity of Alda-1 (benzodioxyldichlororobezamide) to shield the oxidative stress in HMVEC (2).Briefly, HMVEC pretreated with 20 μM of Alda-1 prior to exposure ofhyperoxia show a diminished accumulation of toxic compound 4HNE, asignificant decrease in oxidative stress and a significant decrease inapoptotic activity. Furthermore, Alda-1 pretreated samples in hyperoxiashow improved Aldh2 activity and ALDH2 expression and significantlyenriched mitochondrial membrane potential. Therefore, activation ofALDH2 may inhibit the endothelial dysfunction caused by hyperoxicexposure.

Acute lung injury (ALI) (3) is a serious clinical complication of arespiratory fiasco affecting 200,000 people in US annually (4) with thecases increasing yearly. ALI has caused the death of 70,000 people alonein the USA (5). One type of ALI is acute respiratory distress syndrome(ARDS) of which hyperoxia is an essential part of treatment (6). Routinetreatment involves continuous exposure to hyperoxia however, theafter-effects of hypoxia treatment are injurious to the patient (7).Prolonged exposure to hypoxia causes hyperoxic acute lung injury (HALI)which eventually leads to death as shown in many animal models.Oxidative stress, ROS and apoptosis caused by hyperoxic exposure leadsto dysfunction of endothelial cells (7-9).

HALI has a negligible amount of fibrosis in differentiation whencompared to other lung injury models and hyperoxia helps in mimickingALI (10) (5), thus making focus on the HALI lung injury modelattractive. The hyperoxic exposure generates oxidative stress, speciallypromoting 4HNE, a toxic compound causing harmful effects to mitochondriaand impairing cell transduction, (9, 11). 4HNE is a lipid peroxidationproduct, an ominous reactive aldehyde acting as a biomarker foroxidative stress (8). The oxidative stress-generated 4HNE deploys aharmful impact on a variety of tissues, such as cardiac, neural,epithelial cells, and endothelial cells. (Chen, 2008; Galam, 2015;Neely, 1999; Rahman, 2002; Solito, 2013).

There are various therapies to metabolize oxidative stress, but in lungsALDH2 acts as a therapeutic agent and also a substrate of 4HNE (9,12-15). Another remedy for acute lung injury is to diminish 4HNE and ROSby modification of the molecule's membrane receptor px27 (16) andapoptosis signaling kinase (17). The ALDH2 activator Alda-1 has beenstudied and administration is rarely utilized in experiments (18). Theuse of Alda-1 has been successfully able to enhance the function ofALDH2 and reduce toxic 4HNE effectively in lung ischemia in epithelialcells, cerebral ischemia, cardiac ischemia, and human umbilicalendothelial cells (2, 12, 15, 19)

Until now there have been no studies conducted on human microvascularendothelial cell (HMVEC) in hyperoxia with an Aldh2 activator. The HMVECplay an important role in vascular function and homeostasis in thelungs, and are also involved in remodeling of the vascular wall,angiogenesis, coagulation, blood vessel formation, and acting as aninterface for blood circulation (20). HMVECs are vulnerable to oxidativestress and hemodynamic alteration (7). Hyperoxia-caused oxidative stressis a crucial replica that induces the harmful outcome of ALI byobstructing barriers of endothelial and epithelial cells (Galam, 2016).The long term exposure to hyperoxia causes buildup of ROS and affectscell proliferation, cell toxicity and cell viability (7). Therefore,hyperoxia yields a foundation to probe the pathogenesis of cellulardamage and pulmonary disease.

The effect of Alda-1 was measured on endothelial cells exposed tohyperoxia to evaluate accumulation of 4HNE in hyperoxia. Due tohyperoxia, oxidative stress is increased which leads to apoptosis. Theinventors evaluated the effect of Alda-1 on expression of cytochrome Cand Bax in cells exposed to hyperoxia. An increase of 4HNE in HMVEC wasshown, indicating that mitochondrial function is affected thus theinventors evaluated the effect of Alda-1 on mitochondria formitochondrial membrane potential. Further, the effect of Alda-1 onhyperoxia was measured to evaluate the expression and activity of ALDH2.The results showed that Alda-1 reduced accumulation of 4HNE, oxidativestress, apoptosis and enhanced mitochondrial membrane potential andactivity of ALDH2 in HMVEC cells exposed to hyperoxia. It isparticularly noteworthy that the use of Alda-1 as a treatment in cellsexposed to hyperoxia shows that Alda-1 acts as an agonist to ALDH2 tosuppress the damage caused by oxidative stress that was induced byhyperoxia(12, 15, 18). Alda-1 treatment provides a replacement remedyfor endothelial dysfunction caused by hyperoxic lung injury.

Materials and Methods

Cell Culture

HMVEC's were preserved in EGM-2 MV media (Lonza, Wakersville, Md.) andsupplemented with FBS (25 ml), Hydrocortisone (0.2 ml), hFGF-B (2.0 ml),VEGF (0.5 ml), R3 IGF-1 (0.5 ml), Ascorbic acid (0.5 ml), hEGF (0.5 ml),GA-1000 (0.5 ml) at 37° C. in a 5% carbon dioxide-humidified incubatorto maintain sufficient cell growth. The cultured cells were validatedfor confluence (around 70%) and exposed to hyperoxia for 48 hours withand without Alda-1.

Oxidative Stress Assay

The samples of whole cell lysate (50 μg) were seeded in each well.Oxidative stress was measured by an oxiselect 4HNE adduct competitiveELISA kit (Cell Biolabs), according to the instructions of kit.

Western Blot

The HMVEC-L cells were cultured and after reaching confluence (70%),were exposed to hyperoxia with or without Alda-1 for 48 hours. Theconcentration was determined by the proOX p100 sensor (Biospherix).After hyperoxia, the cell pellets were collected by centrifugation andthe cell pellets were suspended in lysis buffer (20 mM Tris HCL, pH7.4,150 mM Nacl, 0.5% Triton-x 100) and the supernatant was collected aftercentrifugation at high speed (14000 g) for 15 minutes at 4° C. Theamount of protein was calculated by BCA assay kit (Pierce, Rockford,Ill.). Equivalent amount of protein (15 μg) subjected to SDS-PAGE usingbetween 4-20% tris-glycine gel (Bio-rad) after which electro transferwith PVDF membrane was performed, followed by blocking in 5% skim milkwith washes with TBST. The membranes were treated with primary andsecondary antibodies. The bands were developed by pierce EC1 (ThermoFischer scientific) and films with protein bands were scanned andanalyzed densitometrically by NIH ImageJ software. The ratio of proteinto loading control was taken and analyzed in percentages.

JC-1 Staining

HMVEC-L cells were maintained in a EGM-2 (Lonza) media supplemented withFBS (25 ml), Hydrocortisone (0.2 ml), hFGF-B (2.0 ml), VEGF (0.5 ml), R3IGF-1 (0.5 ml), Ascorbic acid (0.5 ml), hEGF (0.5 ml), GA-1000 (0.5 ml)at 37° C. in a 5% carbon dioxide humidified incubator to maintain asufficient cell growth. The HMVEC-L cells were plated at density of1×10,000 cells in a cell view dish with glass bottom (Thomasscientific). When cells attained 70% confluency, they were exposed tohyperoxia for 48 hours with Alda-1 and without Alda-1. Followingexposure to hyperoxia, the HMVEC-L cells were washed by HBSS solution(without Ca+ and Mg+) (Gibco). After the washes, the HMVEC-L weretreated with JC-1 (5 μM) (Thermo Fisher) at 37° C. for 15 minutes,subsequently washed in HBSS solution three times and placed in themedium. The live cell imaging was conducted using fluorescencemicroscopy (Olympus). The green and red images were captured influorescence and images were quantified by image J by red to greenratio.

ALDH2 Activity

The enzymatic activity of ALDH2 was measured by transformation ofacetaldehyde to acetic acid (Chen et al., 2008). The HMVEC-L cells werecultured as mentioned above, followed by 300 μl of buffer (10 mM DTT,100 mM Tris-HCl pH 8.0, 20% glycerol, 1% Triton X-100) and centrifugedat 55000 g for 30 minutes at 4° C. The supernatant was utilized todetect activity of ALDH2 at 340 nm in a spectrophotometer. The assaymixture (1 ml) contained 10 mM NAD+, 100 mM sodium pyrophosphate,acetaldehyde, water, and 100 μg mitochondrial lysate. The assay activitywas started by adding acetaldehyde (10 mM) to the cuvette. Thespecificity of enzyme reaction was expressed as nmol NADH/minute/mgprotein.

Statistical Analysis

All experiments were conducted with 3 samples per group and values wereindicated as means±SE. Statistical significance was calculated by usingMicrosoft Excel with a 2 tailed T-test in which values less than p<0.05were considered as significant.

Results

ALDH2 Activator Attenuates Hyperoxia-Induced 4HNE Accumulation inPulmonary Endothelial Cells

Alda-1 has been shown to protect many different cell types againstoxidative stress by increasing ALDH2 activity. However, no study hasrevealed any similar protective effect of Alda-1 on lung endothelialcells. To verify its anti-oxidative effect on lung endothelial cells,the inventors exposed human microvascular endothelial cells (HMVEC) tohyperoxic conditions in the presence and absence of Alda-1. The resultsshow that pretreatment with Alda-1 attenuates hyperoxia-induced 4HNEincrease in human microvascular endothelial cells. Hyperoxia causes a55% increase in 4HNE compared to normoxia. Pretreatment of HMVEC withAlda-1 in hyperoxic conditions was shown to cause a 26% decrease in 4HNEaccumulation as compared to untreated HMVEC exposed to hyperoxia. Theseresults indicate that ALDH2 plays a vital role in protecting lungvascular endothelial cells from oxidative stress-induced 4HNEupregulation, which is a trigger for cell apoptosis. FIG. 5 shows agraph that represents the whole cell protein lysate for humanmicrovascular endothelial cells (HMVEC) under different conditions inwhich the cells were subjected to ELISA to evaluate the levels of 4HNEcontent during the normoxia, hyperoxia, and hyperoxia+Alda1 (20 μM)treatment for 48 hours.

ALDH2 Activator Attenuates Hyperoxia-Induced Downregulation of OxidativeStress in Pulmonary Endothelial Cells

To determine whether ALDH2 activation by Alda-1 suppresses apoptosissignaling caused by oxidative stress in human microvascular endothelialcells, the protein levels of Bax and cytochrome C in whole cell lysatewere quantified by western blotting. The results demonstrated thathyperoxia causes a 2.5-fold significant increase in cytochrome C levelsand a 1.5-fold increase in BAX levels as compared to normoxia. Thepretreatment of HMVEC with Alda-1 in hyperoxia caused a 64% significantdecrease in cytochrome C and a 25% significant decrease in Bax ascompared to cell exposed to hyperoxia without Alda-1 pretreatment. Theseresults indicate that ALDH2 activation helps to protect humanmicrovascular endothelial cells (HMVEC) from oxidative stress-inducedapoptosis. FIG. 6A shows the western blot analysis of the whole celllysate for HMVEC under different conditions (n=3) to evaluate the levelsof cytochrome C during normoxia, hyperoxia and hyperoxia with Alda-1 (20μM) for 48 hours. Equal amounts of protein were loaded per lane (20 μg).FIG. 6B shows expression of cytochrome C was normalized to β-actin andpresented in arbitrary units. The results are shown in mean±SEM.

ALDH2 Activator does not Promote Hyperoxia-Induced Expression of ALDH2in Pulmonary Endothelial Cells

In the existing literature, Alda-1 treatment has been shown to have noimpact on the expression of ALDH2 expression in in vitro in manydifferent cell types. Moreover, no study has been conducted to revealthe expression of ALDH2 in lung microvascular endothelial cells. Toconfirm the effect of Alda-1 on ALDH2 expression, the protein level ofALDH2 in whole cell lysate was evaluated by western blotting. Theresults suggests that pre-treatment with Alda-1 in hyperoxia has noeffect on ALDH2 expression in hyperoxia treatment. These resultsindicate that pretreatment with an ALDH2 activator does not alter theexpression of ALDH2 during hyperoxia in human microvascular endothelialcells. FIG. 7A is an image that depicts the whole cell lysate for humanmicrovascular endothelial cells under different conditions (n=3)subjected to immunoblot to evaluate the levels of ALDH2 during normoxia,hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalizedagainst β-actin. The values indicate standard error mean (*P<0.05).Equal amounts of protein loaded per lane (20 μg). FIG. 7B is a graphshowing expression of ALDH2 was normalized to β-actin and presented inarbitrary units. The results are shown in mean±SEM.

ALDH2 Activator Promotes Hyperoxia-Induced ALDH2 Activity in PulmonaryEndothelial Cells

To evaluate whether the ALDH2 activator Alda-1 enhances ALDH2 activityduring hyperoxia in human microvascular endothelial cells, themitochondrial extract was isolated and an ALDH2 activity assay wasconducted. The results demonstrated that hyperoxia with Alda-1 treatmentcaused a 45% increase in ALDH2 expression compared to hyperoxia withoutAlda-1 pretreatment. These results indicate that the ALDH2 activatorAlda-1 increases the activity of ALDH2 under hyperoxia in humanmicrovascular endothelial cells. FIG. 8A is an image depicting the wholecell lysate for human microvascular endothelial cells under differentconditions (n=3) subjected to immunoblot to evaluate the levels of Baxduring normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48hours normalized against β-actin. The values indicate standard errormean (*P<0.05). Equal amounts of protein were loaded per lane (20 μg).FIG. 8B is a graph depicting expression of Bax normalized to β-actin andpresented in arbitrary units. The results are shown in mean±SEM.

ALDH2 Activator Promotes the Hyperoxia-Induced Mitochondrial MembranePotential in Pulmonary Endothelial Cells

To evaluate the effect of Alda-1 during hyperoxia on human microvascularendothelial cells, the mitochondrial membrane potential was quantifiedby JC-1. JC-1 is considered to be an indicator of cellular damage andcell death. The cellular damage can be attributed by decrease influorescence as a red to green ratio. The results demonstrated thatmembrane potential exhibited a 1.90-fold increase in normoxia comparedto hyperoxia and a 1.62-fold increase in hyperoxia with Alda-1 treatmentcompared to hyperoxia without Alda-1 treatment. These results suggestthat the ALDH2 activator, Alda-1, decreases cellular damage duringhyperoxia in human microvascular endothelial cells (P<0.005). FIG. 9Ashows the human microvascular endothelial cells under differentconditions subjected to the JC1 staining to evaluate the mitochondrialmembrane potential during normoxia, hyperoxia and hyperoxia with Alda-1(20 μM) for 48 hours. The values indicate standard error mean (*P<0.05).FIG. 9B is a graph depicting Jc1 images quantified using imageJ softwarewith an intensity expressed in arbitrary units. The results are shown inmean±SEM (Magnification=200×).

Hyperoxia exposure causes cellular dysfunction in HMVECs, and prolongedexposure to hyperoxia causes organ failure (7). During hyperoxicexposure, the cell undergoes oxidative stress due to an increase in ROS(21). Although Alda-1 has been shown to reduce injuries in the heart,liver, brain, kidney, and intestines, it has not previously beenthoroughly tested in the lung (12, 22-25). ALDH2 has also beenimplicated in numerous pathologies, such as ischemia, Alzheimer'sdisease, and Parkinson disease. The use of Alda-1 has been proven toattenuate endothelial cell injuries in neural and umbilical cells. Forthe first time, the inventors have demonstrated that the use of Alda-1,an ALDH2 activator, is capable of attenuating endothelial dysfunction inpulmonary human microvascular endothelial cells (HMVECs) duringhyperoxia. Mitochondrial ALDH2 plays a pivotal role in preventing theaccumulation of 4HNE (15, 18), preventing the accumulation of oxidativestress (8), preventing expression of apoptotic marker, and enhancingmitochondrial membrane potential (15, 26).

It has been effectively shown that in vitro exposure to hyperoxia causesan accumulation of the toxic compound 4HNE which causes excessivealveolar protein leaking, and pulmonary edema, both of which contributeto ALI (26). For the first time the inventors provide evidence that 4HNEcauses extensive damage in HMVECs. The inventors pretreated HMVECs withAlda-1, exposed them to 48 hours of hyperoxia and showed that hyperoxicexposure caused an accumulation of 4HNE, but that treatment with Alda-1reduced that amount. Hyperoxic exposure in vivo has been shown toincrease the formation of adducts of 4HNE (27). Moreover, these proteinalterations result in impaired cellular feedback, thereby causing theproduction of ROS and oxidative stress, which ultimately leads tocellular dysfunction and death (8, 16).

The exposure of pulmonary cells to hyperoxia for prolonged durationscauses damage to epithelial and endothelial cell barriers, therebyincreasing ROS, oxidative stress, and apoptosis, and decreasing themitochondrial membrane potential (9, 28). Hyperoxia also causes anincrease in 4HNE levels affecting mitochondrial membrane potential.These changes occur within 48 hours of exposing HMVECs to hyperoxia. Thedisaggregation and death of HMVEC can be seen in hyperoxia (FIG. 5). Theincrease in mitochondrial membrane potential, ALDH2 activity and ALDH2expression of HMVECs pretreated with Alda-1 (FIGS. 9A, 10 and 7Arespectively) shows mitochondrial oxidative stress induced by hyperoxia.

For the first time, the inventors have revealed that, in HMVECs exposedto hyperoxia, ALDH2 activity and ALDH2 expression increase as a resultof the Alda-1 treatment during hyperoxia (FIGS. 7A and 10). When treatedwith Alda-1 prior to the hyperoxia exposure, the recovery of Aldh2decreased cytochrome C release and repaired mitochondrial membranepotential (FIGS. 6A and 9A). Alda-1 provided powerful protection againstthe effects of hyperoxia in HMVECs, as shown by the diminished levels ofBax, an apoptotic activator indicative of mitochondrial stress (FIG.8A). The importance of Alda-1 shielding ALDH2 against these disastrouseffects has been profoundly described by Perez Miller et al.

The inventors demonstrate for the first time the protective effects ofAlda-1 in HMVECs undergoing 48 hours of hyperoxic exposure. Thepretreatment of Alda-1 before hyperoxic exposure caused a decrease in4HNE levels versus HMVECs that were exposed to hyperoxia alone (FIG. 5).This decrease is believed to be because ALDH2, which has increasedactivity in cells treated with Alda-1, converts 4HNE into acetaldehyde,which is then converted into acetic acid by ALDH2 as well (Xu, Guthrieet al. 2006). There was no change in the expression of ALDH2 in thegroup treated with DMSO or Alda-1 prior to hyperoxia exposure (FIG. 7).The hyperoxic ALDH2 expression levels were high when compared tonormoxia because ALDH2 exerts protective effects during hyperoxicexposure, thereby leading to its natural overexpression. Overall, Alda-1was successfully able to enhance the activity of ALDH2 in hyperoxicconditions to relieve oxidative stress. Literature shows that ALDH2 is acritical protein in numerous tissues, such as liver, heart, muscle, andkidney (30), and for the first time the inventors demonstrate itsimportance in lung tissues, namely Human Lung Microvascular Endothelialcells.

Conclusion

The inventors have shown that hyperoxic damage can be guarded by ALDH2activation via pretreatment with Alda-1. For the first time the effectsof Alda-1 in human microvascular endothelial cells during hyperoxia havebeen shown. It has been shown that treatment with Alda-1 diminishes theaccumulation of 4HNE and reduces oxidative stress by decreasing ROSduring hyperoxia. Hyperoxia has been shown to elevate oxidative stress,depolarize the mitochondrial membrane, decrease ALDH2 activity, disruptcellular transduction, and cause apoptosis. The use of Alda-1 reversedthe harmful effects of hyperoxic injury to HMVECs, revealing thatoxidative stress is important in the pathological processes of variousdiseases associated with the lung and endothelial cells. Therefore,administration of Alda-1 may act as a novel therapeutic drug to mitigatehyperoxic injury in lungs.

Example 4—Alda-1 Treatment Maintains Mitochondrial Homeostasis andReduces Immune Cell Infiltration In Vivo

Acute respiratory distress syndrome (ARDS) is associated with fluidfilled lungs and hypoxia. Accordingly, ARDS patients are placed onsupplemental oxygen; however, hyperoxia can damage the lungs, causemitochondrial dysfunction, and ultimately lead to acute lung injury inanimal models of ARDS. Mitochondrial aldehyde dehydrogenase 2 (ALDH2)metabolizes dangerous, reactive products, such as 4-hydroxynonenal(4HNE), that otherwise causes oxidative stress. The inventors examinedwhether Alda-1, an ALDH2 activator, enhances the activity of ALDH2, thusalleviating hyperoxia damage by preventing mitochondrial dysfunction andreducing immune cell infiltration. The inventors also examined ifadministration of Alda-1 recovers mitochondrial dynamics and reducescytokine levels and immune cell infiltration.

Methods

C57BL/6 mice were exposed to hyperoxia for 48 hours with DMSO (Control)and Alda-1 (20 mM) via alzet pumps on the dorsal side of mice. The micewere divided into four groups: normoxia+DMSO (room air),normoxia+Alda-1, hyperoxia+DMSO, and hyperoxia+Alda-1. The lungs wereharvested and the bronchoalveolar lavage (BAL) fluid was collected andanalyzed by immunoblot and cytokine levels.

Results

Immunoblot analysis of lung lysate demonstrated that mice treated withAlda-1, compared to mice treated with DMSO, during hyperoxia havedecreased levels of OPA1 and Drp1, indicating that Alda-1 shieldsagainst oxidative stress for proteins associated with mitochondrialdynamics. Moreover, the BAL fluid analysis reveals less infiltration ofmacrophages in mice treated with Alda-1 versus mice treated with DMSOalone. These results indicate that activation of ALDH2 via Alda-1 isprotective against hyperoxia-induced acute lung injury and may be aviable therapeutic agent for ARDS.

Conclusion

These findings suggest that Alda-1, an ALDH2 activator, maintainsmitochondrial homeostasis and reduces immune cell infiltration in thismouse model of ARDS and may be a novel therapeutic agent in ARDS.

Example 5—Alda-1 Reduces the Effects of ALI Induced by Hyperoxia byProtecting Mitochondrial Dynamics In Vivo

Acute Lung Injury (ALI) is characterized by acute and severeinflammation of the lungs that can result in respiratory failure. Themain symptom of ALI is shortness of breath associated with low oxygen.It is characterized by X-ray findings of bilateral pulmonaryinfiltrates. The most common treatment for ALI is to providesupplemental oxygen, which can lead to the accumulation of reactiveoxygen species (ROS) and the toxic metabolite 4-hydroxy-2-noneal (4HNE).This leads to further oxidative stress, thus limiting cell proliferationand triggering aberrations in mitochondrial dynamics. Mitochondrialdysfunction is one of the hallmarks of ALI. Mitochondrial Aldehydedehydrogenase 2 (ALDH2) acts as a mitochondrial shield against damage.Activation of ALHD2 via Alda-1 also can attenuate the mitochondrialdamage during hyperoxic exposure. This example is focused on thetherapeutic potential of Alda-1 to reduce the effects of hyperoxicexposure in ALI by protecting mitochondrial dynamics.

Methods

C57BL/6 mice were pretreated with DMSO (control) and 20 μM Alda-1(administered via Alzet pumps), then exposed to hyperoxia for 48 hours.The mice were divided into three groups: Room air, hyperoxia (48hour)+DMSO, and hyperoxia (48 hour)+Alda-1. The lung tissues wereharvested, and lysates evaluated by immunoblot analysis.

Results

Western blot analysis of lung lysates indicates that mice treated withAlda-1 during hyperoxia versus mice treated only with hyperoxia havedecreased levels of mitochondrial fusion proteins mitofusin 1 (MFN1) andmitofusin 2 (MFN2) relative to mice that were not treated. In addition,mitophagy protein PTEN-induced kinase 1 (PINK1) levels also decrease inmice treated with Alda-1 prior to hyperoxia exposure compared tohyperoxic controls. These data suggest that Alda-1 protects mitochondriaagainst hyperoxia-induced damage by maintaining mitochondrialhomeostasis.

Conclusion

These findings suggest that Alda-1 may be a treatment option to regulatethe mitochondrial dynamics via activation of the mitochondrial enzymeAldehyde dehydrogenase 2 (ALDH2).

Example 6—Alda-1 Reduces the Effects of ALI In Vivo

Acute lung injury (ALI) is a critical lung disorder where theinefficient oxygen uptake causes acute hypoxemia and Acute RespiratoryDistress Syndrome (ARDS). There are nearly 200,000 annual cases of ALIin the United States alone, and the incidence rate is increasing. At themolecular level, hyperoxic exposure causes an increase in reactiveoxygen species, leading to an accumulation of 4-Hydroxy-2-nonenal(4HNE), a lipid peroxidation product causes protein adducts and inhibitsthe activity of the mitochondrial enzyme ALDH2 (Aldehyde dehydrogenase),which is involved in metabolism of alcohol. Use of Alda-1 may reduce theeffect of acute lung injury.

Methods

The C57BL/6 mice were embedded with osmotic pumps and continuouslyinjected with either DMSO or Alda-1 (20 μM), then they were exposed to100% oxygen 48 hours. The lung samples, as well as Bronchial AlveolarLavage Fluid, were collected for the evaluation of infiltration ofcytokines, inflammation, autophagy, and apoptosis by Diff Kwik staining,H&E staining, and western blot.

Results

The mice treated with DMSO in hyperoxia showed more cytokine andneutrophil infiltration and elevated inflammation than mice treated withAlda-1 in hyperoxia. The western blot analysis of Alda-1-treated miceshowed reduced cytochrome C release, reduced LC3B, and reducedNF-κB-p65, while also showing a decrease in oxidative stress,inflammation and autophagy.

The findings imply that Alda-1, an ALDH2 activator is a potentialtherapeutic drug for the treatment of acute lung injury.

Conclusion

The inventors have shown, both in vitro and in vivo, that hyperoxicdamage can be prevented and treated by administration of Alda-1.Treatment of endothelial dysfunction in pulmonary HMVECs due tohyperoxia, as well as in mice exposed to hyperoxia, with Alda-1diminishes the accumulation of 4HNE and reduces oxidative stress bydecreasing ROS during hyperoxia. Treatment with Alda-1 reversed theharmful effects of hyperoxic injury and enhanced the mitochondrialmembrane, increased ALDH2 activity, and decreased apoptosis. Therefore,administration of Alda-1 acts as a novel therapeutic drug to mitigatehyperoxic injury in lungs.

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The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. While the disclosure issusceptible to various modifications and implementation in alternativeforms, specific embodiments have been shown by way of non-limitingexample in the drawings and have been described in detail herein. Sincecertain changes may be made in the above construction without departingfrom the scope of the instant application, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned

The disclosure is not intended to be limited to the particular formsdisclosed. Rather, the disclosure is to cover all modifications,equivalents, and alternatives falling within the scope of the disclosureas defined by the following appended claims and their legal equivalents.

Without further elaboration, it is believed that one skilled in the artcan use the preceding description to utilize the present disclosure toits fullest extent. The examples and embodiments disclosed herein are tobe construed as merely illustrative and exemplary and not a limitationof the scope of the present disclosure in any way. It will be apparentto those having skill in the art, and having the benefit of thisdisclosure, that changes may be made to the details of theabove-described embodiments without departing from the underlyingprinciples of the disclosure herein.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A method of preventing hyperoxic acute lunginjury in pulmonary cells comprising: administering a therapeuticallyeffective amount of a composition to the pulmonary cells comprising atherapeutically effective amount of Alda-1; and a pharmaceuticallyacceptable excipient; wherein the composition is administered prior tohyperoxic exposure; wherein the therapeutically effective amount of thecomposition containing the Alda-1 reduces damage to the pulmonary cellscaused by oxidative stress due to hyperoxic exposure to preventhyperoxic acute lung injury.
 2. The method of claim 1, wherein theadministration of the therapeutically effective amount of thecomposition containing the Alda-1 enhances mitochondrial membranepotential in the pulmonary cells.
 3. The method of claim 1, wherein theadministration of the therapeutically effective amount of thecomposition containing the Alda-1 suppresses mitochondrial reactiveoxygen species (ROS) production in the pulmonary cells.
 4. The method ofclaim 1, wherein the administration of the therapeutically effectiveamount of the composition containing the Alda-1 decreases apoptosis ofthe pulmonary cells.
 5. The method of claim 1, wherein theadministration of the therapeutically effective amount of thecomposition containing the Alda-1 decreases accumulation of4-hydroxy-2-noneal (4HNE) in the pulmonary cells.
 6. A method ofpreventing hyperoxic acute lung injury in pulmonary cells comprising:administering a therapeutically effective amount of a composition to thepulmonary cells comprising a therapeutically effective amount of Alda-1;and a pharmaceutically acceptable excipient; wherein the composition isadministered during hyperoxic exposure; wherein the therapeuticallyeffective amount of the composition containing the Alda-1 reduces damageto pulmonary cells caused by oxidative stress due to hyperoxic exposureto prevent hyperoxic acute lung injury.
 7. The method of claim 6,wherein the administration of the therapeutically effective amount ofthe composition containing the Alda-1 enhances mitochondrial membranepotential in the pulmonary cells.
 8. The method of claim 6, wherein theadministration of the therapeutically effective amount of thecomposition containing the Alda-1 suppresses mitochondrial reactiveoxygen species (ROS) production in the pulmonary cells.
 9. The method ofclaim 6, wherein the administration of the therapeutically effectiveamount of the composition containing the Alda-1 decreases apoptosis ofthe pulmonary cells.
 10. The method of claim 6, wherein theadministration of the therapeutically effective amount of thecomposition containing the Alda-1 decreases accumulation of4-hydroxy-2-noneal (4HNE) in the pulmonary cells.